The main goal of this research is to understand how the spinal cord generates locomotion. This involves identifying the essential components of the locomotor network, recording their activity patterns and isolating the critical cellular and synaptic properties responsible for locomotor network function. We have recently begun using optogenetics to investigate the function of several different neuronal classes in the neonatal spinal cord. These include cholinergic neurons, neurons expressing the islet-1 transcription factor, neurons expressing the en-1 transcription factor, glia expressing S100-B or GFAP. In collaboration with Dr. A. Lev-Tov (Hebrew University, Israel), we have identified a novel class of sacral commissural interneuron whose axons travel in the ventral funiculus. These neurons appear to mediate, in part, the excitation of lumbosacral locomotor networks by stimulation of afferents in the sacrocaudal cord. We now propose to identify their connections, transmitter phenotype and function during locomotor-like activity. We are also proposing an ambitious set of experiments to image the activity of all (or most) of the neurons and glia during a single cycle of locomotor activity in a hemi-segment of the spinal cord. This information will allow us to establish how neurons are recruited during each cycle of activity and how variable the recruitment patterns are from cycle to cycle and from animal to animal. Moreover, it will enable us to determine if the different methods for inducing locomotor-like activity (drug-induced, brainstem stimulation, dorsal/ventral root stimulation) all activate the same networks. In addition, we will exploit the growing list of mouse cell lines, in which various interneuron classes have been marked with GFP or one of its variants, to identify the activity patterns of identified interneurons. Finally, we are using calcium and voltage sensitive dye imaging to identify the activity patterns of motoneurons during locomotion of the nematode worm C.elegans. C. Elegans is a simple worm in which calcium and voltage sensitive dyes can be expressed in the identified neurons of motor circuits. Because of its rapid generation time compared to mice, this organism can serve as a test bed for introducing genes into specific sets of neurons and evaluating their utility as probes of neural activity. In addition, the neural mechanisms that underlie movement in this animal will provide specific hypotheses that can be tested in the much more complex nervous systems of mice.